Biochip and the production method thereof

ABSTRACT

The invention relates to a biochip comprising a substrate presenting a reflecting main surface and wherein the main surface presents localized sites that are made sensitive to fluorescence detection by a transparent layer having optical thickness (2k+1)λ/4 where k is a positive or zero integer and where λ designates a wavelength lying between a wavelength λ 0  at which fluorescence is excited and a wavelength λ 1  at which fluorescence is emitted. The biochip can be manufactured by depositing a transparent layer of thickness mλ′/2 on a substrate and by making studs or wells of thickness (2k+1)λ/4 in said layer to form said sensitive sites.

The present invention relates to a biochip of the type enablingbiological or biochemical probes to be detected by fluorescence, and theinvention also relates to a method of manufacturing it.

BACKGROUND OF THE INVENTION

Biochips are very high performance analysis tools in fields relating tomolecular biology. The technology for preparing them is in the processof becoming established. Their use will enable research to beaccelerated and will lead, in particular, to new diagnostic techniques.

A biochip is a set of systems of biological recognition substances suchas biological molecules, all operating in parallel. Amongst biochips,those which have been developed to the greatest extent so far are DNAchips and there are great hopes that they will enable recognitionefforts to be greatly increase in the field of molecular genetics.

Physically, biochips are structures constituted by a solid supporthaving distinct zones distributed thereon, in general of microscopicdimensions, and supporting identical biological recognition probesubstances, each zone or site having a single type of substance whichmay be of biological or biochemical origin. Recognition is performed bya specific affinity interaction between the probe substance fixed on thesupport and the target substance contained in the solution to beanalyzed. The biochip is read by marking the target substance using amarker, generally a fluorescent marker. During recognition, the targetsubstance brings its marker to the zone that supports the probesubstance. Knowing the nature of the probe makes it possible to know thetarget substance with which the affinity reaction has taken place.

OBJECTS AND SUMMARY OF THE INVENTION

The invention seeks firstly to improve the technical quality of thebiochip, and secondly to improve the quality with which the fluorescencecan be read.

The fixing of molecules such as oligonucleotides or strands of DNA onthe support in order to make the biochip relies on chemical reactionsthat take place at the surface of the solid. Usually, the probebiological molecule for fixing is in solution. Depending on the relativesurface properties of the support and of the solution containing theprobes to be fixed, the deposited solution may tend to spread out overthe substrate, making it impossible to obtain zones of interest that areof controlled size and small dimensions.

Similarly, direct localized synthesis of biological molecules such asoligonucleotides can be performed by localized deposition on the surfaceof the substrate of a portion of the reagents, and in particularphosphoramidites. These various reagents are usually in solution in asolvent such as acetonitrile. This solvent has the property of beinghighly wetting, which means that if the necessary precautions are nottaken it causes the reagents to be spread over the surface. Thus, theseproperties prevent the area on which synthesis takes place to becontrolled and thus prevents the zones of interest from being definedgeometrically.

In addition, analysis is performed by putting the biochip into contactwith a solution containing the target molecules which are marked beforeor after contact with a fluorescent group (or possibly they are markedin radioactive manner). Recognition is the result of relatively strongbiochemical coupling between the probe and the analyte (e.g. by forminghydrogen bonds). The analyte is thus fixed on the recognition area andcan be identified by knowledge of the probe that is coupled or securedto said area or site.

In parallel with this specific recognition, depending on the nature ofthe support, target molecules can also become fixed in non-selectivemanner thereon (usually by an adsorption process). This parasiticphenomenon has the effect of reducing the signal-to-noise ratio whenreading.

An object of the present invention is to provide a solution to thoseproblems in order to improve the biological quality of the chip byacting on its optical quality.

In addition, in an advantageous variant, the improvement in thephysicochemical quality of the chip by localized doublefunctionalization can enable the site supporting the biological probemolecules to be better defined geometrically while still ensuring thatall of the sites are identical in size and are in alignment, and whileconsiderably reducing non-selective adsorption of target molecules.

The invention thus provides a biochip comprising a substrate presentinga reflecting main surface, wherein the main surface presents localizedsites that are made sensitive to fluorescence detection by a transparentlayer having optical thickness (2k+1)λ/4 where k is a positive or zerointeger and where λ designates a wavelength lying in the range from awavelength λ₀ at which fluorescence is excited and to a wavelength λ₁ atwhich fluorescence is emitted. The term “optical thickness” is used tomean the product of the actual physical thickness multiplied by therefractive index of the transparent layer.

In the biochip, the main surface presents sites that have been madesensitive to fluorescence detection by a transparent layer of opticalthickness equal to (2k+1)λ₁/4 where k is a positive integer or zero,thereby enhancing fluorescent emission.

In the biochip, outside the sensitive sites, the main surface is coveredin a transparent layer of thickness mλ′/2 where m is a positive integeror zero and λ′ designates a wavelength lying in the range λ_(o) to λ₁.

Preferably, outside the sensitive sites, the main surface is covered ina transparent layer of thickness mλ₀/2, thus serving to preventfluorescence being excited.

In an advantageous embodiment that makes use of localized silanization,and outside the sensitive sites, the surface of the substrate is coveredin a first thin layer, in particular a monolayer of molecules of a firstsubstance A, comprising a hydrocarbon chain of the (CH₂)p type with1<p<30 carrying a catching-hold group such as a silane or a silanol atone end enabling a covalent bond to be made, and carrying a chemicalfunction at its other end that is stable and inert, e.g. CH₃ andhalogenated derivatives thereof. Said first substance is hydrophobic.

In a preferred embodiment, the sensitive sites are covered in a secondthin layer, in particular a monolayer of molecules of a second substanceB presenting a group at one end suitable for fixing to the substrate bymeans of a covalent bond, e.g. a silane function or a silanol function,and presenting a group at its other end suitable for fixing in covalentmanner to a probe molecule, e.g. a group presenting the acid COOHfunction or the alcohol OH function.

Said second substance is hydrophilic.

In the biochip, the surface of the substrate is covered in a layer ofsaid substance B, and outside the sensitive sites, said layer is coveredin a stop layer C.

The invention also provides a method of manufacturing a biochip asdefined above, the method implementing the following steps:

a) depositing a transparent layer of thickness mλ′/2 on a substrate; and

b) making wells or studs in said transparent layer in which thethickness of the transparent layer is equal to (2k+1)λ/4, so as to formsaid sensitive sites.

Depending on the values of k and m, it is possible to make sensitivesites that are in the form of wells or of studs.

When the sensitive zones are wells, the method may implement thefollowing steps:

c) localized double silanization by selectively depositing said firstthin layer, said deposition optionally being followed by heat treatment;and

d) immersing the substrate in a solution containing the second substanceB.

When the sensitive zones are studs, the method implements the followingsteps:

c′) localized double silanization by selectively depositing substance Bon the studs, said deposition optionally being followed by heattreatment; and

d′) immersing the support in a solution containing the substance A.

In another embodiment, the localized double silanization is performed asfollows (after b):

e) a layer of substance B is deposited over the entire surface of thesubstrate, e.g. by immersion in the solution containing substance B. Thefunction for fixing the probe substance is then deprotected andactivated.

f) A monolayer of molecules of a stop substance C is then locallydeposited on the surface in such a manner as to leave the sensitivezones 5 bare. The molecules of the substance C have at one end afunction which reacts with the activated function of the substance B.The other end of the molecule has a group that is highly inertchemically, such as a methyl group or indeed a fluorine-substitutedmethyl group. Thus, only activated sensitive zones are suitable forfixing the probe substances, the outside surfaces of the sensitive zonesbeing made definitively inert against catching hold of biologicalsubstances.

Between the functional groups of the molecule of the substance C, theremay exist an organic chain such as an aliphatic chain of the (CH₂)q typewhere 0<q<30.

It is important to observe that the functionalization layers are ofthicknesses of the order of 1 nanometer (nm) to 10 nm and thereforepossess optical thickness that is negligible compared with thetransparent layer and thus has no direct action on the opticalproperties of the biochip.

Optionally, steps c) and/or d), and/or e), and/or f), and/or g), and/orh) may be followed by heat treatment for facilitating silanization.

BRIEF DESCRIPTION OF THE DRAWINGS

Other characteristics and advantages of the invention will appear betteron reading the following description given with reference to theaccompanying drawing in which FIGS. 1 a to 1 e show a first embodimentof the method of manufacture. A preferred embodiment is shown in FIGS. 1a to 1 d and in FIGS. 2 a to 2 c. A variant is shown in FIG. 3.

MORE DETAILED DESCRIPTION

U.S. Pat. No. 6,008,892 describes a structure that takes advantage ofthe optical properties of thin plates: when a transparent layer ofoptical thickness equal to one-fourth of the wavelength or an oddmultiple of one-fourth of the wavelength lies on a reflecting (orpartially reflecting) substrate, then light emitted by a fluorescentsource emitting at this wavelength is at a maximum. In that method, theentire surface of the substrate is treated uniformly so as to increaseemission by fluorescence. Parasitic fluorescence due to biologicalsubstances adsorbed outside the sensitive zones is thus improved in thesame manner as the fluorescence in the sensitive zones, therebyachieving no improvement in the signal-to-noise ratio between thesensitive zones and the non-sensitive zones.

The present invention remedies that drawback by structuring thethickness of the transparent layer of the substrate, thereby enablingthe fluorescence of sensitive zones to be improved while on the contraryinhibiting fluorescence outside the sensitive zones.

In the invention, the optimized structure is made from a layer 10 havingan optical thickness λ/2 (or more generally mλ/2), being made of atransparent material deposited on the surface 6 of a substrate 1 thatreflects at the reading wavelength, for example a layer 10 of silica orof silicon nitride on a silicon substrate, or a layer of metallic oxideon a metal substrate, e.g. TiO₂ on titanium, ZrO₂ on zirconium, etc.(FIG. 1 a). A film 7 of photosensitive resin is deposited on this layer10 (FIG. 1 b). The resin is exposed through a mask 3 which enablesopening 4 to be opened in the resin 2 after it has been developed, theopenings serving to form sensitive sites on the chip (FIG. 1 c).Chemical etching is then performed to reach an optical thickness for thefilm that is equal to (2k+1)λ/4 (FIG. 1 d) in the localized zones 12 soas to form wells 5 constituting the sensitive sites of the chip.

Preferably, the emission of fluorescence in the sensitive sites isenhanced by selecting a transparent layer of optical thickness(2k+1)λ₁/4. Excitation of fluorescence is preferentially inhibitedoutside the sensitive sites by selecting a transparent layer having anoptical thickness of mλ₀/2.

After the resin 2 has been eliminated, a film of a silane 15 having atits end a functional group suitable for immobilizing the biologicalmolecule can advantageously be deposited in the wells 5 (FIG. 1 e). Thestructure is then raised in temperature in order to enhance thesilanization reaction. The thickness of this thin layer (generallymonomolecular) does not significantly change the optical properties ofthe structure.

Thereafter, probe biological molecules are fixed on the varioussensitive sites by ex situ or in situ techniques. The free top surfaceof the layer is subjected finally to treatment having the effect ofneutralizing the active functional groups that have not reacted duringthe preceding stage. If molecules should nevertheless become fixedoutside the sensitive zone by the deposition solution overflowing, forexample, then the fluorescence signal outside the sensitive zone isweaker by a factor of about 200 than the signal coming from within thesensitive zone and, in most cases, it will have no effect on reading.

Thus, the optical path difference between the inside and the outside ofthe wells improves the signal-to-noise ratio when reading fluorescenceby minimizing the fluorescence signal outside the wells and byoptimizing the fluorescence signal inside the wells.

It is possible to improve both the biochip by simultaneously improvingboth the optical quality and the physicochemical quality of thesubstrate.

The substrate is micromachined by chemical etching so as to make thesensitive sites in the layer as described above (FIGS. 1 a to 1 d). Atransfer operation is then performed.

After chemical etching and eliminating the resin (FIG. 1 d), the chip isput into contact with a transfer surface 30 having a fine layer 21 of asolvent deposited thereon, the solvent containing a substance of type A,e.g. a hydrophobic silane (FIG. 2 a). The hydrophobic silane is thustransferred onto the top surface of the chip as to form a thin layer 22(FIG. 2 b). Silane can also be deposited by an inking device using thesilane-containing solution. Heat treatment encourages the silanizationreaction to take place on the surface of the chip. A second silanizationstep is undertaken, for example by dipping the chip in a solutioncontaining the second silane, and serves to silanize a thin layer 23 onthe bottoms of the initially bare wells (the second silane does not fixto the first) (FIG. 2 c). The specific second silane has at its free enda functional group of a kind that is known per se and that makes itpossible either to achieve a covalent bond with the biological substancein order to hold said biological substance in place, or else to start insitu synthesis of the probe biological molecule: this starting reactionalso corresponds to establishing a covalent bond.

The thickness of the thin layers 22 and 23 (no more than a few layers ofmolecules) is not sufficient to modify significantly the opticalproperties of the surface, which thus combines the selective propertiesobtained by the two techniques.

The method thus makes use of physicochemical confinement. It consists indefining zones that are to receive probes of a hydrophilic nature, bylocally using microtransfer to deposit molecules for forming ahydrophobic monolayer in the region between the sensitive sites. Theinsides of the sensitive sites are then silanized using a silanesuitable for holding onto previously synthesized probes or for in situsynthesis of biological molecules. The microtransfer of the hydrophobicmonolayer is performed by using a transfer surface 30 which may beplane, or which may be constituted by a micropad presenting transferregions in relief.

The transfer surface 30 is put into contact with a solution containingthe hydrophobic silane. A fine film 22 of solution is then transferredby contact at localized positions on the layer 10 of the substrate 1.The substrate 1 is then raised to a temperature of about 100° C. inorder to facilitate the silanization reaction. By using a specificprotocol and selecting an appropriate molecule, it is possible to obtaina monolayer that is organized and compact, known as a self-assembledmonolayer (SAM). Zones that have not received the silane are thentreated so as to be covered in another silane whose free end possesses afunctional group that enables in situ synthesis of oligonucleotides tobe performed (e.g. a hydroxyl group, or that enables biologicalmolecules that have been presynthesized or that are of biological originto be held in place by using an appropriate functional group (e.g. anacid). The grafting reaction of the second silane takes place on thesilanol groups of the silica or the glass constituting the substrate butit cannot take place on the monolayer of the fixed first silane. Thus,only those zones that are to act as sensitive sites are functionalized.

In addition, the hydrophobic silane limits non-selective adsorption verystrongly, and as a result very significantly improves thesignal-to-noise ratio when reading by fluorescence (or possibly byradioactive marker), by increasing the ratio of the true signal in zonessupporting biological probes to any signal that might come from barezones where non-selective adsorption might have occurred.

In general, it is possible to perform the same function using substancesother than silane.

It is possible to deposit a physically and chemically inert layer on thetop portion of the support outside the sensitive site, thereby limitingadsorption and fixing of probe biomolecules while making biochips andlimiting the adsorption or fixing of target biomolecules duringbiological analysis, thereby improving the measurement signal-to-noiseratio.

This inert layer is constituted by a molecule comprising a hydrocarbonchain of the (CH₂)p type with 1<p<30 or a polymer chain, e.g.poly-ethylene-glycol, etc. or an assembly of various chain portions,carrying at one end a group that enables a covalent bond to be formedwith the support, e.g. a silane or a silanol bond, while the other endcarries a chemical function that is stable and inert such as CH₃ or itshalogenated derivatives substituting all or some of the hydrogens in thealiphatic chain (CH₂)p and the CH₃. The layer that is formed may bemonomolecular or polymeric depending on the nature of the first terminalgroup. The layer may advantageously be deposited by contact with a planesurface previously covered in a solution of said molecule.

Thereafter a monolayer of molecules presenting different functions ateach end should be deposited on the sensitive sites: one serves to fixthe molecule by a covalent bond on the substrate, e.g. a silanolfunction, and the other serves to fix the probe biomolecule in covalentmanner, e.g. the acid function COOH or an alcohol function OH. Thismonolayer then serves to hold biomolecules that have been presynthesizedor that are of natural origin, or indeed it enables the probebiomolecules (oligonucleotides, PNA, protein, etc.) to be synthesized insitu in the wells.

Another method is described with reference to FIG. 3.

The surface of the substrate is covered in a layer 30 of the substanceB, then a layer 31 of the stop substance C is deposited locally outsidethe sensitive site, in this case the wells 5.

Preferably, the sensitive sites are made to be effective in detection byusing two techniques that have complementary effects: the physicaleffect associated with the difference in height between the insides ofthe sites and the outside, and the physicochemical effect associatedwith the surface property differences between the insides of the sitesand the outside. As a result, the shape and the relative position of thesensitive sites obtained by a microtechnological type technique are welldefined. This contributes to obtaining a high quality chip and improvingthe reading thereof.

Finally, by associating two techniques, three-dimensionally structuringthe support and preparing its surface to enable attachment to take placein preferred manner in the sensitive sites, e.g. by double silanization,it is possible to improve the quality of the chip and to optimizereading thereof by acting on four aspects which can be implementedseparately or together:

-   -   improving the geometrical definition of the chip by using        masking and/or transfer techniques;    -   decreasing the size of the sensitive sites by the combined        effects of the physicochemical confinement and the machining of        the sites;    -   decreasing non-specific adsorption on the zone between the sites        by depositing a monolayer of hydrophobic silane by        microtransfer, for example; and    -   improving the optical quality with which the chip is read by        optimizing the thicknesses of the transparent support layer.

1. A biochip comprising a substrate containing a reflecting mainsurface, wherein the main surface presents localized sites covered by atransparent layer to enhance fluorescence, whereby the sites are madesensitive to fluorescence detection of fluorescent light emitted bytarget molecules marked by a fluorescent material that is deposited overthe transparent layer, wherein the transparent layer has an opticalthickness of (2k+1)λ/4 where k is a positive integer or zero and where λdesignates a wavelength lying in the range from a wavelength λ₀ at whichfluorescence of the fluorescent material is excited to a wavelength λ₁at which the fluorescence of the fluorescent material is emitted, andfurther wherein, outside the sensitive sites, the main surface of thesubstrate is covered in a transparent layer of thickness different thanthat of the transparent layer of the sensitive sites, the thicknessoutside the sensitive sites being of mλ′/2 where m is a positive integerand λ′ designates a wavelength lying in the range λ₀ to λ₁.
 2. A biochipaccording to claim 1, wherein the transparent layer covering the siteshas an optical thickness equal to (2k+1)λ₁/4 where k is a positiveinteger.
 3. A biochip according to claim 1, wherein, outside thesensitive sites, the main surface of the substrate is covered in atransparent layer of thickness mλ′/2 where m is a positive integer andλ′ designates a wavelength lying in the range λ_(o) to λ₁.
 4. A biochipaccording to claim 3, wherein, outside the sensitive sites, the mainsurface of the substrate is covered in a transparent layer of thicknessmλ₁/2.
 5. A biochip according to claim 1, wherein, outside the sensitivesites, the surface of the substrate is covered in a thin layer ofmolecules of a first substance A comprising a hydrocarbon chain of the(CH₂)p type with 1<p<30 carrying a catching-hold group at one endenabling a covalent bond to be made with the substrate, and carrying achemical functional group at its other end that is stable and inert. 6.A biochip according to claim 5, wherein the thin layer of molecules of afirst substance A is a monolayer of molecules.
 7. A biochip according toclaim 5, wherein the catching-hold group is a silane or a silanol.
 8. Abiochip according to claim 5, wherein the chemical functional group isCH₃ or halogenated derivatives of CH₃.
 9. A biochip according to claim7, wherein the first substance comprises a hydrophobic silane.
 10. Abiochip according to claim 1, wherein the sensitive sites are covered ina thin layer of molecules of a second substance B presenting a group atone end suitable for fixing to the substrate by means of a covalent bondand presenting a group at its other end suitable for fixing in covalentmanner to a probe molecule.
 11. A biochip according to claim 10, whereinthe thin layer of a second substance B is a monolayer of molecules. 12.A biochip according to claim 10, wherein the group suitable for fixingto the substrate by means of a covalent bond is a silane functionalgroup or a silanol functional group.
 13. A biochip according to claim10, wherein the group suitable for fixing to a probe molecule is a grouppresenting an acid COOH functional group or an alcohol OH functionalgroup.
 14. A biochip according to claim 12, wherein said secondsubstance is a hydrophilic silane.
 15. A biochip according to claim 10,wherein the surface of the substrate is covered in a layer of saidsecond substance B, and wherein, outside the sensitive sites, said layerof said second substance B is covered in a stop substance C.
 16. Amethod of manufacturing the biochip of claim 1, the method comprisingthe following steps: a) depositing a transparent layer of thicknessmλ′/2 on a substrate, where m is a positive integer or zero and λ′designates a wavelength lying in the range λ₀ to λ₁; and b) making wellsor studs in said transparent layer in which the thickness of thetransparent layer is equal to (2k+1)λ/4, where k is a positive integeror zero and where λ designates a wavelength lying in the range from awavelength λ₀ at which fluorescence is excited and to a wavelength λ₁ atwhich fluorescence is emitted, so as to form said sensitive sites.
 17. Amethod according to claim 16, further comprising the following steps: c)depositing said first thin layer of a first substance A comprising ahydrocarbon chain of the (CH₂)p type with 1<p<30 carrying acatching-hold group at one end enabling a covalent bond to be made withthe substrate, and carrying a chemical functional group at its other endthat is stable and inert by selective transfer onto the substrate; andd) immersing the substrate in a solution containing a second substance Bpresenting a group at one end suitable for fixing to the substrate bymeans of a covalent bond and presenting a group at its other endsuitable for fixing in covalent manner to a probe molecule.
 18. A methodaccording to claim 17, wherein step c) and/or d) is followed by heatingthe substrate to a temperature effective to facilitate the formation ofa covalent bond.
 19. A method according to claim 16, wherein, after stepb), the method further comprises the following steps: c) depositing athin layer of a substance B presenting a group at one end suitable forfixing to the substrate by means of a covalent bond and presenting agroup at its other end suitable for fixing in covalent manner to a probemolecule, over the entire surface of the substrate; and d) depositing alayer of a stop substance C outside said sensitive sites.
 20. A biochipaccording to claim 1, wherein k is zero and, outside the sensitivesites, the main surface of the substrate is covered in a transparentlayer of thickness mλ′/2 where m is a positive integer and λ′ designatesa wavelength lying in the range λ₀ to λ₁.